Sequencing Matters: Comparative Effectiveness of Density-Gradient Centrifugation and MACS for Enhancing Sperm Quality and DNA Integrity

Background: Sperm DNA fragmentation (SDF) is increasingly recognized as a clinically relevant marker of male reproductive potential and has been associated with impaired embryo development, miscarriage, and suboptimal assisted reproductive technology outcomes, particularly in selected infertile populations. Because conventional sperm-preparation methods do not directly target molecular sperm damage, combined selection strategies such as density gradient centrifugation (DGC) and annexin V-based magnetic-activated cell sorting (MACS) have been proposed to enrich spermatozoa with improved DNA integrity before intracytoplasmic sperm injection (ICSI). Regardless of their more favorable functional characteristics, the optimal sequence for combining these methods remains uncertain. Methods: This study presents a hybrid analysis combining a structured review of 25 published studies with original prospective observational laboratory data. The review examined the effects of DGC, MACS, and sequential combined protocols on SDF, conventional semen parameters, and reported assisted reproduction outcomes, with particular attention to the unresolved question of sequencing order. In parallel, a blinded observational cohort of 67 infertile men with baseline SDF of at least 30% underwent sequential DGC followed by MACS, with SDF measured before and after processing using the sperm chromatin dispersion test (Halosperm ® assay). Results: Across the reviewed literature, combined DGC/MACS approaches generally reduced SDF more than single-step preparation, although the magnitude of benefit varied across assays, patient populations, and laboratory protocols. Some studies also reported improvements in embryo quality and pregnancy-related outcomes, but these data were derived mainly from retrospective or small comparative cohorts, and no large randomized head-to-head trials were identified comparing DGC→MACS with MACS→DGC for fertilization, miscarriage, or live-birth outcomes. In the observational cohort, DGC→MACS was associated with a significant mean absolute SDF reduction of 19.42% +/− 9.4% (p < 0.0001). Most patients (85.1%) achieved post-treatment SDF values below the 30% threshold. Conclusions: Combined DGC/MACS sperm preparation, supported by the data in our study sequence, appears promising for reducing SDF in selected infertile men, particularly those with elevated baseline SDF. However, the optimal sequencing strategy and its impact on major reproductive endpoints remain uncertain and require better standardized comparative clinical studies.